Preparation of live vaccines

ABSTRACT

Described is a method for the generation of a live vaccine containing stable bacteria carrying at least three attenuating mutations and a vaccine containing bacteria obtained by said method.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 14/440,942, filed 6 May 2015, which is a nationalphase application pursuant to 35 USC § 371 of International patentapplication number: PCT/EP2013/068373, filed on 5 Sep. 2013, whichclaims benefit of priority to European patent application no.12008206.0, filed on 7 Dec. 2012; all of which are incorporated byreference in their entirety.

DESCRIPTION

The present invention provides a method for the generation of a livevaccine containing stable bacteria carrying at least three attenuatingmutations and a vaccine containing bacteria obtained by said method.

Many of the live bacterial vaccines comprise attenuated bacteria thathave been manipulated by biomolecular techniques. Unfortunately, most ofthese vaccines are considered as being insufficient to comply with therequirements of practice for the following reasons:

(a) The production is complex and time consuming, the degree ofattenuation cannot be controlled and, accordingly, adaptation to thesusceptibility of the host is often unsatisfactory.

(b) Methods of testing (clinical trials) requested by the legislativeauthority are also elaborate.

(c) The population to be vaccinated is limited.

By contrast, mutants attenuated by metabolic drift (MD) arecharacterized by the following advantages:

(a) Costs for preparation are low and the degree of attenuation via thedesired selection of an increased generation time and, thus, reducedcolony size, respectively, is, in principle, almost arbitrary.

(b) When using stable specific vaccine strains having three attenuatedMD mutations for vaccination of farm animals, elaborate methods oftesting are not required.

(c) Even smaller lot sizes will pay off.

As regards key data of the evolutionary principle of MD attenuation thefollowing should be stressed:

(a) The interplay of pathogenic agent versus host is based on mutualtolerance. Highly susceptible hosts survive as single individuals whenaccidentally infected by an attenuated mutant of a highly virulentpathogenic agent. The host population rejuvenates via the few survivingindividuals. The pathogenic agent proliferates as adapted attenuatedstrain. Myxomatosis is a typical example of such process. Conclusion:Bacterial populations (as well as populations of fungi and viruses)always contain gradually attenuated mutants, inter alia so-called MDmutants.

(b) MD mutants represent clones having mutations in metaboliccompartments per definitionem resulting in dysfunction (i.e.attenuation=fitness cost). As a consequence, gradually reduced colonysizes (depending on the clone) compared to the wild strain can be found.Normally, these mutants are eliminated by the immuno competent host or,alternatively, are overgrown by the adapted normal flora.

(c) The reduced colony sizes of the MD mutants inversely correlate withthe (prolonged) generation time and the (increasing) degree ofattenuation.

(d) The convincing efficacy of MD attenuated test vaccines and vaccineshas been proven.

MD mutants can be selected and isolated as:

(a) spontaneous MD antibiotic resistence (MD “res”) clones of, e.g.,streptomycin, rifampicin, fosfomycin, fusidic acid, and nalidixic acid.These clones can be isolated with a frequency of more than 1% inrelation to the virulent resistant clones. MD “res” and virulentresistant clones result from different mutations. Accordingly, MD “res”and attenuation can be regarded as a functional entity.

(b) Increased environmental stress tolerance (iet) mutants whichindirectly accumulate in the “dying off” culture.

(c) streptomycin independent (Sm-id) suppressor mutants derived fromstreptomycin dependent (Smd) clones. These two marker mutants consist ofa broad spectrum of clones characterized by clone specifically graduatedreduced colony sizes and increasing degrees of attenuation,respectively, from almost wild type virulence to over-attenuation (minicolonies). Generally, ribosomal mutations increase the normal misreading(mistranslation) more or less and the exclusive suppressor mutation alsocauses attenuation.

For immunization of, e.g., populations of chicken with live Salmonellaand Campylobacter vaccines the interruption of the chain of infection tohuman beings and, as a consequence, the reduction of human enteritidesis the primary goal. Normally, chicks tolerate facultative pathogenicSalmonella (and generally even Campylobacter) without showing anyclinical symptoms. Thus, the low virulence of these wild strains forchicks requires vaccine strains showing a moderate degree of attenuationensuring on the one hand immunogenicity for chicks but excluding on theother hand a hazard for human beings.

One criterion of the efficacy of vaccine strains is, e.g., theverifiable reduction of the degree of colonization after challenge. MDattenuated live vaccines expressing all components of the bacteria(e.g., outer membrane proteins) can be regarded as a practice orientedoption, even as regards Campylobacter.

Thus, it is possible to develop effective vaccines for facultativepathogenic bacteria such as Salmonella and Campylobacter, providedover-attenuation is avoided by adjusting to a low or moderate degree ofattenuation. In other words, the reduction of colony size as attenuationequivalent should not fall below about 25% of the colony size of thewild strain. In addition, this condition sine qua must be in line withthe safety requirements of the WHO:

stability due to the presence of two independent attenuating mutations.The reversion rate per marker is about 10⁻⁸. However, there is a needfor developing vaccine strains showing even higher stability, i.e.,lower reversion rates, but not over-attenuation. Unfortunately, theintroduction of additional mutations to increase the safety of a livevaccine usually leads to an excess of attenuation thereby rendering thevaccine less effective.

Thus, the technical problem underlying the present invention is toprovide improved live vaccine strains characterized by increasedstability.

The solution of said technical problem is achieved by providing theembodiments characterized in the claims, i.e., to provide improved livevaccine strains with an increased stability based on at least threemutations, yet avoiding over-attenuation and allowing for the adjustmentof the attenuation to a desired level. In fact, during the experimentsleading to the present invention it could be shown that by use of the MDattenuation vaccine strains characterized by three (or even more)independent attenuating mutations can be generated showing increasedstability and a degree of attenuation that does not exceed the degree ofattenuation of vaccine strains having two MD “res” attenuatingmutations. In the experiments of the present invention streptomycin wasused, however the procedures disclosed in the present invention are notrestricted to only this antibiotic. The experiments described below arebased on the use of so-called Sm-id clones derived from Smd mutantssince these “double marker” mutants (comprising clones showing alldegrees of attenuation—from wild strain-like colonies to mini colonies)allow for the generation of vaccine strains showing a degree ofattenuation corresponding to the degree of attenuation of MD “res”single marker strains. Strains of the present invention (Sm-id/MD “res”)show an increased stability of about 10⁻²⁴.

So far, Sm-id/MD “res” vaccine strains of Salmonella and Campylobacterhave not been described in the prior art. In addition, vaccine strainshaving four, five or even six attenuating mutations generated by thegraduated incorporation of two or three Sm-id mutations and anadditional MD “res” (A a, A a) marker are novel as well, forstreptomycin: Smd 1→Sm-id I→Smd a→Sm-id II→Sm-α→Sm-id III (sixattenuating mutations) and Sm-id I/Sm-id II/MD “res” (five attenuatingmutations), respectively.

It was found that the particular spectrum of revertants—graduatedlyreduced colony sizes—of the Sm-id mutants with combinations of two, fouror six markers starts: (a) for Sm-id strains with colony sizes of thewild strain, (b) for Sm-id I/Sm-id II strains with colony sizes of Sm-idI-like colony sizes and (c) for Sm-id I/Sm-id II/Sm-id III constructsthe largest colony found only corresponded to Sm-id It Sm-id II-likeclones. Thus, these are not wild strain-like revertants.

In principle, the selection and isolation of Smd mutants as startingstrains for the generation of Sm-id clones belongs to the state of theart. About 10% of the normal Sm-resistant mutant colonies of, e.g.,Staphyloccoci, Escherichia coli, Bacillus cereus, are Smd clones (as anapparent biological principle). However, this does not apply toSalmonella and Campylobacter. The frequency of the appearance of Smdclones of Salmonella among the colonies of mutants showing resistance isreported to be about 0.1% or the isolation of such strains is onlyachievable by use of mutagenesis, respectively.

Interestingly, after analysis of about 5000 Sm resistant mutants thepresent inventors could not find any Smd strains. In addition, it has tobe stressed that the spectrum of Sm-id revertants varies depending onthe Smd clone, accordingly, several Smd clones are needed as startingmutants. In other words, the common procedure for selection isinapplicable.

Since presumably ribosomal Smd-pathways are an evolutionary principle ofadaptation two possible mechanisms were tested:

(a) Smd-mutants enter the death phase immediately after their formation.Thus, such mutants can only be isolated from log-phase cultures, e.g.,from ≤18 h/37° C. cultures but not from ≥48 h/37° C.F cultures

(b) During status nascendi the Smd clones are fragile, show delayedgrowth as mini colonies (which might be overlooked). These mini coloniesadapt to “normal growth” during passaging.

For Campylobacter there are no data available as regards Smd- and Sm-idmutants. In the literature, there are only some hints as regardsSm-resistant mutants: clones showing high resistance could only beobtained by use of multiple passages against increasing concentrationsof streptomycin. A one-step Sm-resistance could only be achieved by useof a concentration of 20 μg streptomycin/ml. Experiments of the presentinventors for isolating mutants showing Sm-resistance and Smd-mutantsusing 100 μg streptomycin/ml (by plating of 72 h/39° C. cultures)failed. Surprisingly, it was found that despite significantly increasingamounts of bacteria the viable counts significantly decreased from the24 h/39° C. to the 72 h/39° C. culture as shown in the following table.

TABLE 1 Campylobacter coli and Campylobacter jejuni: Viable counts onPetri dishes with Caso-agar against the incubation period at 39° C.Campylobacter 24 h 48 h 72 h C. coil 1 × 10¹⁰ 3 × 10⁹ 7 × 10⁸ C. jejuni3 × 10¹⁰ 5 × 10⁹ 1 × 10⁹ C. coli Smd 3 × 10⁹ 3 × 10⁸ 4 × 10⁷

These results are in line with the observation that cultures ofCampylobacter are capable of passing into a viable but not culturablestatus.

Accordingly, mutants having Sm resistance could only be obtained byplating of ≤24 h/39° C. culture material. However, no Smd mutants couldbe found among the resistant colonies having normal size or slightlyreduced size. But among the very small colonies and mini colonies ofCampylobacter (appearing with distinct delay) the majority of the clonescould be characterized as being Smd clones.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show examples of Salmonella vaccine strains characterized bythree attenuating mutations.

Incubation of the plates was at 37° C. for about 20 hours and resultedin the following:

FIG. 1A Salmonella infantis (wild strain/Sm-id 4.22/Rif 2 (and Iet 4));

FIG. 1B Salmonella virchow (wild strain/Sm-id 9.3/Rif 2);

FIG. 1C Salmonella hadar (wild strain/Sm-id 4.1/Sm 2 (and Rif 1);

FIG. 1D Salmonella paratyphi B, variant Java 3.2 (wildstrain/Sm-id.1.1/Rif 3 (and Rif 2)).

FIGS. 2A-2D show examples of Campylobacter vaccine strains characterizedby three attenuating mutations Incubation of the plates was at 39° C.for about 48 and 72 hours, respectively and resulted in the following:

FIG. 2A Campylobacter coli I (wild strain/Sm-id 5.5/Pho 1 and Pho 2);

FIG. 2B Campylobacter coli II (wild strain/Sm-id 18.1/Sm 2);

FIG. 2C Campylobacter jejuni I (wild strain/Sm-id 2.3/Pho 1 (and Pho2));

FIG. 2D Campylobacter jejuni II (wild strain/Sm-id 2.1/Sm 2).

FIGS. 3A-3B show examples of Salmonella vaccine constructs characterizedby (two), four, five or six attenuating mutations. Incubation of theplates was at 37° C. for about 20 hours and resulted in the following:

FIG. 3A Salmonella virchow:

wild strain/Sm-id I 1.4/Sm-id II 0.3/Sm 4;

wild strain/Sm-id I 1.4/Sm-id II 0.3/Sm-id III 0.x.

FIG. 3B Salmonella infantis:

wild strain/Sm-id I 4.22/Sm-id II 0.1/Rif 2;

wild strain/Sm-id I 4.22/Sm-id II 0.1/Sm-id III 1.1.

FIGS. 4A-4B show examples of Campylobacter vaccine constructscharacterized by (two), four, five or six attenuating mutations.Incubation of the plates was at 39° C. for about 48 hours and resultedin the following:

FIG. 4A Campylobacter coli II wild strain/Sm-id I 18.1/Sm-id II a.1.This strain is characterized in that the second Sm-id a.1 mutationresults in an Sm resistance.

FIG. 4B Campylobacter coli II wild strain/Sm-id I 17.7/Sm-id IIa.1/Phol, wild strain/Sm-id I 17.7/Sm-id II a.1/Sm-id III a.1.

Thus, the present invention provides a method for the generation of abacterial live vaccine containing stable bacteria carrying at leastthree (and up to six or seven) attenuating mutations, wherein saidmethod comprises the following steps:

(a) providing a bacterial strain and growing said strain in the presenceof a first antibiotic, preferably streptomycin;

(b) isolating from the strain of (a) such “mini” colonies whichcorrespond to clones which are dependent on the first antibiotic;

(c) growing a clone of (b) in the absence of the first antibiotic andisolating attenuated revertants characterized by a colony size which is≥50% of the colony size of the wild strain;

(d) growing a clone obtained in step (c) in a medium supplemented with asecond antibiotic that may differ from the first antibiotic (e.g., anaminoglycoside such as streptomycin, neomycin, kanamycin, spectinomycin,gentamicin, amikacin, and tobramycin; rifampicin, fusidic acid,nalidixic acid, fosfomycin,) having a suitable concentration, preferablyan about tenfold MIC;

(e) isolating and serially passaging colonies showing reduced size (MD A“res”); and

(f) isolating clones having the graduated reduction of the colony sizeas stable property.

The bacterial strain of step (a) is, preferably, obtained from “wild”virulent strains. These strains can be taken from diseased animals(e.g., chicken). The starting natural strains which are used should havea certain degree of virulence.

The choice of the antibiotic for selecting the mutants of step (a) isguided by reasons of a practical nature. For example, streptomycin isknown to lead rapidly to the development of resistant and dependentstrains among the micro-organisms.

Thus, in a preferred embodiment of the method of the present inventionthe antibiotic of step (a) is streptomycin. However, otheraminoglycoside antibiotics such as neomycin, kanamycin, spectinomycin,gentamicin, amikacin, and tobramycin, and rifampicin, fusidic acid andnalidixic acid may also be suitable as the antibiotic of step (a).

It is known that resistance to the antibiotic can result from differentmodification mechanisms. In particular, the genetic modification mayaffect a chromosome of the bacterium. The chromosomal modification is arare event which, once carried out, ensures the stability of theacquired properties.

The term “mini colonies” as used herein relates to bacterial coloniescharacterized by a reduction of size. Preferably, they are characterizedby a size of ≤10% of the corresponding wild strain colonies.

The selection of attenuated bacterial strains as a function of growthcriteria on media containing an antibiotic is an operation which hasbeen used for various species with the object, notably, of causing theappearance of strains having reduced virulence.

The bacterial strains according to the invention characterized by atleast three (and up to six or seven) attenuating mutations are in thefirst place non-virulent strains selected, from natural virulentstrains, for their growth capacity on a medium with a high content of anantibiotic such as streptomycin, and in addition, which can only bedeveloped satisfactorily in the presence of the antibiotic (step (b).For this reason, these strains are said to be dependent on, e.g.,streptomycin (Smd mutant).

In the second place, the strains of step (c) are mutants selected fromthe antibiotic dependent strains and which have the particularity ofbeing able to develop in the absence of streptomycin due to theintroduction of a second attenuating mutation or marker. These strainsare called Sm-id strains. Preferably, a washing step is carried outbetween steps (b) and (c). The preferred medium for step (c) is aSalmonella Caso (SC) medium (e.g., for Salmonella) or a Caso medium(e.g., for Campylobacter).

Step (d) allows introducing an additional MD antibiotic resistance(“res”) mutation (as a third attenuating marker). Preferably, theantibiotic is streptomycin, rifampicin or fosfomycin. The concentrationof the antibiotic in step (d) can be determined by the person skilled inthe art according to routine procedures. Preferably, for Salmonella theconcentration of rifampicin, streptomycin (note: most Sm-id mutants arestreptomycin sensitive and therefore suited for an additional MD Sm“res” marker) and fosfomycin corresponds to an MIC value of abouttenfold, and for fusidic acid to an MIC value of about fourfold.Preferably, for Campylobacter, at least 200 μg fosfomycin/ml and atleast 100 μg streptomycin/ml, respectively, are used.

In step (e) the Sm-id/MD antibiotic “res” strains of the previous stepcharacterized by an additional reduced slight colony size are isolatedand serially passaged in order to check stability. Preferably, at least30 serial passages are carried out.

Finally, in step (f) the clones isolated from step (e) having thegraduated reduction of the colony size as stable property are provided.

In a preferred embodiment of the method of the invention steps (a) to(c) are at least repeated once for the generation of bacteria carryingat least four attenuating mutations. This method allows to generate newattenuating mutants, e.g., according to the following schemes (shown forSm):

(a) Smd 1→Sm-id I→Smd a→Sm-id II;

(b) Smd 1→Sm-id I→Smd a→Sm-id II→Smd α→Sm-id III;

(c) Smd 1→Sm-id I→Smd a→Sm-id II→Smd α→Sm-id III→MD antibiotic “res”.

Surprisingly, it was found that strains derived from Sm-id mutantscarrying four or even six mutations do not show a higher degree ofattenuation, compared to the strains having three attenuating mutations,but an even higher stability.

The choice of the antibiotic for selecting the MD antibiotic “res”mutant strains according to the present invention is guided by reasonsof a practical nature and, in principle, any antibiotic capable ofinducing metabolic drift (MD) mutations can be used for the purposes ofthe present invention, e.g., streptomycin (note: most Sm-id mutants arestreptomycin sensitive and therefore suited for an additional MD Sm“res” marker), rifampicin, fosfomycin, fusidic acid or nalidixic acid.

The method of the present invention is not restricted to particularbacteria. Besides Salmonella sp. and Campylobacter sp., other bacteriasuch as Staphylococcus aureus, Escherichia coli, Bacillus cereus(Pseudoanthrax), Yersinia sp. such as Y. pestis, Klebsiella sp.,Listeria sp., Aeromonas sp., Shigella sp., Pasteurella/Avibacterium sp.,Riemerell a sp., Ornithobacterium rhinotracheale, Bordetella sp., andPseudomonas sp. can also be used for generating bacterial live vaccinecontaining stable bacteria according to the methods of the presentinvention.

However, preferred bacteria are Salmonella and/or Campylobacter,especially Salmonella bongori, the S. enterica subspecies enterica,arizonae, diarizonae, salamae, houtenae and indica, preferably S.enterica subspecies enterica such as the following Serovars: Dublin,gallinarum (biovars gallinarum and pullorum), choleraesuis, Typhisuis,typhi, Paratyphi A,B,C, Abortusequi, Abortusovis, Abony, Enteritidis,Typhimurium, Copenhagen, infantis, virchow, hadar, agona, Newport,anatum, Heidelberg, panama, indiana, Saintpaul, Brandenburg, andCampylobacter coli, Campylobacter jejuni, and Campylobacter fetus.

In a preferred embodiment of the method of the present invention insteps (a) and (b) Salmonella mutants are isolated from log phasecultures and as mini-colonies that start appearing after at least ormore than 48 h at 37° C. incubation.

In a further preferred embodiment of the method of the present inventionin steps (a) and (b) Campylobacter mutants are isolated as mini-coloniesthat start appearing after at least or more than 72 h at 39° C.incubation.

The present invention also provides alive bacterial strains obtainableby the method of the invention as well as a vaccine comprising alivebacterial strains of the invention and a biologically acceptablecarrier. The vaccinating compositions may of course be constituted bymeans of freshly cultivated bacteria.

Preferably, the vaccine composition of the present invention isfreeze-dried.

To administer the vaccinating bacteria, the medium in which they aresuspended is not critical. Of course, this medium must not interferewith the good viability of the bacteria that they contain.

The vaccine of the present invention is administered in an amountsuitable for immunization of an individual and may additionally containone or more common auxiliary agents. The employed term “amount suitablefor immunization of an individual” comprises any amount of bacteria withwhich an individual can be immunized. An “amount suitable forimmunization of an individual” may be determined using methods known toone skilled in the art. The term “individual” as used herein comprisesan individual of any kind. Examples of such individuals are animals (andhumans).

The administration of the vaccine preferable is the oral route but alsoinjection may be made at various sites of the individualintramuscularly, subcutaneously, intradermally or in any other form ofapplication. It may also be favourable to carry out one or more “boosterinjections” having about equal amounts.

The vaccine of the present invention may be prophylactic, that is, thecompounds are administered to prevent or delay the development of aninfection or colonisation, e.g. an infection/colonisation caused bySalmonella or Campylobacter.

The following strains have been deposited with the German Type CultureCollection (Deutsche Sammlung von 25 Mikrorganismen and Zellkulturen(DSMZ), Braunschweig) on Nov. 27, 2012 under the Budapest Treaty:

Name Accession Number Salmonella enterica ssp. enterica DSM 26682Serovar Infantis Smid4-22/Rif2 = Campylobacter coli K2848/11 Smid18/Sm2= DSM 26683 Campylobacter jejuni K2963/12 Smid2.1/Sm2 = DSM 26684

The below examples explain the invention in more detail.

Example 1 Materials

(A) Strains

Salmonella enterica subsp. enterica serovar virchow,

Salmonella enterica subsp. enterica serovar infantis,

Salmonella enterica subsp. enterica serovar hadar,

Salmonella paratyphi B (var. L-Tartrat+, formerly Java),

Campylobacter coli, Campylobacter jejuni (provided by Lohmann AnimalHealth, Cuxhaven, Germany).

(B) Media

1000 ml Campylobacter medium (Caso-medium) contain: 35 g Caso Agar(Sifin), 3 g yeast extract, 3 g casein hydrolysate, 4 g activatedcarbon, 0.25 g FeSO₄, 0.25 g sodium pyruvate, 5 g agar Kobe (Roth).

1000 ml Salmonella medium (SC-medium) contain: 35 g Caso Agar (Sifin), 3g yeast extract, 1 g glucose, 5 g agar Kobe (Roth).

(C) Antibiotics

Streptomycin (Sm) (Roth No. 0236.2), fosfomycin (Pho) (Sigma No. P5396),rifampicin (Rif) (Riemser Arzneimittel AG, Fatol Eremfat 600 mg)

(D) MIC Values of Wild Type Strains

Strain Streptomycin rifampicin fosfomycin Salmonella enterica 12.5 12.5n.d. subsp. enterica serovar Virchow Salmonella enterica 12.5 12.5 n.d.subsp. enterica serovar Infantis Salmonella enterica 25 12.5 n.d. subsp.enterica serovar Hadar Salmonella paratyphi B 30 12.5 n.d. (var.L-tartrate+) Campylobacter coli WS I 1 n.d. 25 Campylobacter coli WS II1 n.d. 25 Campylobacter jejuni WS I 2 n.d. 25 Campylobacter jejuni WS II2 n.d. 25 n.d.: not determined

Example 2 Selection and Isolation, of Smd Mutants

(a) Practice-Orientated Isolation of Smd Mutants of Salmonella

About 10¹⁰ cfu of a 18 h/37° C. culture of Salmonella were plated on aPetri dish containing SC agar supplemented with 500 μg streptomycin/ml.Besides colonies having normal sizes and single colonies having slightlydecreased sizes (virulent Sm resistant clones and MD Sm “res” clones)“mini colonies” (predominantly small colony variants=scv) with varyingfrequencies—depending on the strain—could be detected. After anincubation time of about ≥48 h (at 37° C.) 1 to 2 additional minicolonies (per about 30 colonies having normal sizes and colonies havingslightly decreased sizes) could be detected that could not bedistinguished from scv. Depending on the frequency of appearance of thescv phenotype 3% to 20% of these mini colonies could be shown torepresent Smd mutants.

The calculated frequency of the Smd clones in relation to resistantmutants was ≥1%.

Note: The isolation of Smd clones is achieved by use of Sm sensitivewild type strains as the starting material. Strains preferably have alow MIC value.

(b) Practice-Orientated Isolation of Smd Mutants of Campylobacter

Bacterial material obtained from a Caso agar Petri dish culture (24h/39° C.; about 10¹⁰ cfu) that had been inoculated in such a way thatthe entire surface of the disc was covered was plated on 1 or 2 Casoagar Petri dishes supplemented with 100 μg streptomycin/ml and incubatedfor 72 h at 39° C. Depending on the strain ≤10 colonies/plate (averagevalue) having normal sizes and colonies having slightly decreased sizes(streptomycin resistant and MD Sm “res” clones) were detectable. Inaddition, colonies having a clearly reduced size (diameter is ≤25% ofthe normal size) with a frequency of about 20%—compared to the colonieshaving normal sizes and colonies having slightly reduced sizes—could bedetected. About one-third of these colonies were Smd clones.

The calculated frequency of the Smd clones in relation to resistantmutants was 5%.

Example 3 Selection and Isolation of Sm-Id Mutants

(a) Isolation of Salmonella Sm-Id Mutants from Smd Clones

About 10⁹ cfu (per petri dish) of a washed Smd mutant were plated withSC medium and incubated for 48 h at 37° C. From the attenuatedrevertants obtained only such mutants were further treated that showed acolony size of about 50% compared to the wild type strain colonies(according to the objective to obtain Sm-id clones having only lowattenuation).

(b) Isolation of Campylobacter Sm-Id Mutants from Smd Clones

Bacterial material obtained from a Caso agar (supplemented with 100 μgstreptomycin/ml) Petri dish culture (24 h/39° C.) that had beeninoculated in such a way that the entire surface of the disc was coveredwas subjected to one washing step, plated on Caso medium in a ratio of1:1 (about 3×10⁹ cfu) to 1:4 and then incubated for 72 h at 39° C. Underthese culturing conditions the majority of Smd clones showed thedevelopment of ≤10 attenuated revertants (on average). Most of theseattenuated revertants were Sm sensitive. Generally, Sm-id clones showinga reduced colony size of about ≥50% compared to the wild type straincolonies were further processed.

Some strains, e.g., Campylobacter jejuni, allowed isolation only fromSm-id having a colony size of ≤50% compared to the wild type strain.

Note: Not all Campylobacter strains and the Smd mutants derived thereofallow isolation of Sm-id revertants without any problems. However, it ispossible to isolate Sm-id revertants also from the problematic strainsusing, for example, several independent Smd mutants.

Example 4 Isolation of an Additional MD Antibiotic “Res” Mutant

The incorporation of an additional MD antibiotic “res” mutation inselected Sm-id mutants as third marker for attenuation and recognitionwas carried out as already described above.

Briefly,

(a) Salmonella: 10⁹⁻¹⁰ cfu of the selected Sm-id clones were incubatedon SC medium supplemented with an about tenfold MIC value concentrationof rifampicin or streptomycin (as regards fusidine acid the aboutfourfold MIC value concentration), respectively, and incubated for 48hours at 37° C.

(b) Campylobacter: The material of a Petri dish culture (Caso medium)that was inoculated with the Sm-id mutant in such a way that it coveredthe whole surface and incubated for 24 h at 39° C. was plated at a ratioof 1:4 to 1:8 on Caso medium supplemented with 200 μg fosomycin/ml or100 μg streptomycin/ml and incubated for ≥72 h at 39° C.

The colonies showing (more or less) reduced sizes were isolated andsubjected to serial passages. About 20% of these clones maintained theclone specifically graded reduction of colony size as a stabile feature.

Example 5 Generation of Vaccine Strains Having 4 or 6 AttenuatedMutations

The generation of vaccine strains having 4 or 6 attenuated mutations wasachieved by sequentially incorporating a second and, optionally, a thirdSm-id suppressor mutation into a basic Sm-id I clone: Sm-id I/Sm-idII/Sm-id III.

(a) Salmonella: About 10¹⁰ cfu of the basic Sm-id I mutant (or the Sm-idII starting strain) were plated on SC medium supplemented with 500 μgstreptomycin/ml and incubated for 48 h at 37° C. About 5% ofSm-resistant colonies are Smd mutants (now growing primarily as colonieshaving “normal sizes”). By use of these Smd clones derived from Sm-id Istrains and Sm-id II strains, respectively, Sm-id mutants were againisolated according to the approach described in Example 3a. Cloneshaving the desired reduction of colony size were treated further.

(b) Campylobacter: The material obtained from a Caso medium Petri dishculture that was inoculated with an Sm-id mutant in such a way that theentire surface was covered and incubated for 24 h at 39° C. was platedat a ratio of 1:4 on Caso medium supplemented with 100 μgstreptomycin/ml and incubated for 72 h at 39° C. Besides the about 15 Smresistant colonies having a “normal size” 2 to 3 small colonies could bedetected. 50% of these colonies are Smd clones. These Smd clones(derived from Sm-id I strains and Sm-id II strains, respectively) wereused as starting clones—according to Example 3(b)—for again isolatingSm-id mutants. Clones showing the desired reduction of colony size weretreated further.

Example 6

Isolation of an MD Antibiotic “Res” Mutant from Selected Sm-Id IIMutants

(a) Salmonella: The incorporation of an advantageous MD antibiotic “res”mutation into selected Sm-id If Sm-id II mutants as an additional 5^(th)attenuation- and recognition marker was carried out analogouslyaccording to the approach described in Example 4(a).

(b) Campylobacter: The incorporation of an advantageous MD antibiotic“res” mutation into selected Sm-id I/Sm-id II mutants as an additional5^(th) attenuation- and recognition marker was carried oat analogouslyaccording to the approach described in Example 4(b).

Note: The approach described above can also be used for the additionalincorporation of an MD antibiotic “res” mutation into selected Sm-id IIImutants (having six attenuated mutations) as 7^(th) marker forattenuation and recognition. However, this might result inover-attenuation, which might interfere with relevancy to practice.

Example 7 Colony Sizes Converted to Bar Graphs for ProspectivelyOriented Evaluation of the Probable Degree of Attenuation

Suspensions of the corresponding wild type strains and the MD mutantsderived from these strains are diluted logarithmically and then platedon culture medium in such a way that per Petri dish 10 to 50 welldefinable single colonies can be obtained. At least 5 Petri dishes pergrade of dilution are prepared in order to compensate for differences ingrowth due to the medium. Single colonies grown under standardizedconditions (e.g., identical times of incubation, identical layerthicknesses of the medium) are photographed. Digital photographs areprocessed with the CellProfiler program (Broad Institute): The diametersof the individual colonies were determined and saved. After averaging ofthe values the data are plotted as bar graphs in relation to the sizesof the wild type strain colonies (given as 100%).

Example 8

Preparation of Vaccines from Suitable Vaccine Strains and Use forVaccination of Chicks/Chicken and Further Hosts to be Protected,Respectively

For the preparation of live vaccines vaccine strains harbouring three(four, five and six, respectively) attenuating mutations were grown incommon liquid media up to logarithmic phase. Vaccine suspensions andvaccine sediments, respectively, were supplemented with a suitablestabilisator and subsequently lyophilized. The vaccines obtained wereadministrated (according to the kind of indication one, two or threedoses) by oral or parenteral administration.

1. A method for the generation of a stable attenuated bacterial straincarrying three to seven attenuating mutations, wherein said methodcomprises the following steps: (a) obtaining a starting naturalbacterial strain and growing said strain in the presence of a firstantibiotic; isolating from the strain of (a) colonies that are dependenton the first antibiotic and have a size of ≤10% as compared to a colonyof a corresponding starting natural bacterial strain provided in step(a), wherein mini colony is characterized by colonies having a size of≤10% as compared to a colony of a corresponding starting naturalbacterial strain; (b) growing a clone of (b) in the absence of the firstantibiotic and isolating attenuated revertants characterized by a colonysize which is ≥50% as compared to a colony of a corresponding startingnatural bacterial strain provided in step (a); (c) growing a cloneobtained in step (c) in a medium supplemented with a second antibiotic;(d) isolating and serially passaging colonies to identify clones stablybeing of reduced size but greater than 25% in size as compared to acolony of a corresponding starting natural bacterial strain provided instep (a); and (e) isolating clones of step (e) having reduced colonysize as a stable property; wherein the first antibiotic is selected fromstreptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin,tobramycin, rifampicin, fusidic acid, and nalidixic acid; and whereinthe second antibiotic is selected from streptomycin, neomycin,kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin,fusidic acid, nalidixic acid, and fosfomycin; to provide a stableattenuated bacterial strain carrying three to seven attenuatingmutations; and wherein colony size is characterized by the diameter ofthe colony.
 2. The method of claim 1, wherein the first and secondantibiotic is selected from streptomycin, neomycin, kanamycin,spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidicacid, and nalidixic acid.
 3. The method of claim 1, wherein the firstantibiotic is streptomycin, and wherein the second antibiotic isrifampicin.
 4. The method of claim 1, wherein the bacterial strain isselected from the group consisting of Aeromonas species (sp.), Bacilluscereus, Bordetella sp., Campylobacter sp., Escherichia coli, Klebsiellasp., Listeria sp., Ornithobacterium rhinotracheale,Pasteurella/Avibacterium sp., Pseudomonas sp., Riemerella sp.,Salmonella sp., Shigella sp., Staphylococcus aureus, and Yersinia sp. 5.The method of claim 1, wherein the bacterial strain is Salmonella orCampylobacter.
 6. The method of claim 5, wherein Salmonella mutantsselected in steps (a) and (b) are isolated from log phase cultures andas mini-colonies that start appearing after at least or more than 48 hat 37° C. incubation.
 7. The method of claim 5, wherein Campylobactermutants selected in steps (a) and (b) are isolated as mini-colonies thatstart appearing after at least or more than 72 h at 39° C. incubation.8. The method of claim 1, wherein the bacterial strain carries at leastfour attenuating mutations, and steps (a) to (c) are repeated at leastonce.
 9. The method of claim 1, wherein serially passaging comprises atleast 30 serial passages.
 10. The method of claim 1, wherein the secondantibiotic of step (d) comprises a concentration of at least fourfoldMIC and up to tenfold MIC.
 11. The method of claim 1, wherein the secondantibiotic is rifampicin, streptomycin, or fosfomycin and comprises aconcentration of tenfold MIC.
 12. The method of claim 1, wherein thesecond antibiotic is fusidic acid and comprises a concentration offourfold MIC.
 13. The method of claim 1, wherein the bacterial strain isSalmonella, the first antibiotic is streptomycin, and the secondantibiotic is rifampicin.
 14. The method of claim 1, wherein the stableattenuated bacterial strain comprises a degree of attenuation that doesnot exceed the degree of attenuation of vaccine strains having twometabolic drift attenuating mutations.
 15. The Salmonella entericastrain deposited with the German Type Culture Collection (DeutscheSammlung von Mikrorganismen und Zellkulturen (DSMZ)) on Nov. 27, 2012under accession number DSM
 26682. 16. A vaccine comprising theSalmonella strain of claim
 15. 17. The Campylobacter strains depositedwith the German Type Culture Collection (Deutsche Sammlung vonMikrorganismen und Zellkulturen (DSMZ)) on Nov. 27, 2012 under accessionnumbers DSM 26683 or DSM
 26684. 18. A vaccine comprising one or morestrains according to claim 17.